1.1 Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions related to improved methods of producing biologically-active, soluble eukaryotic disulfide bond-containing eukaryotic polypeptides in bacterial cells. In preferred embodiments, disulfide-bond containing eukaryotic fusion proteins such as tissue plasminogen activator (tPA) and pancreatic trypsin inhibitor (PTI) are produced in recombinant transformed Escherichia coli cells using recombinant vector systems which direct the co-expression of the eukaryotic protein and a eukaryotic foldase, such as protein disulfide isomerase (PDI).
1.2 Description of the Related Art
1.2.1 PROTEIN EXPRESSION IN BACTERIAL HOSTS
A significant achievement in molecular biology has been the use of recombinant bacterial cells to produce eukaryotic proteins. This method has been particularly useful for production of medically important polypeptides that are obtained in low yield from natural sources. Often otherwise difficult to obtain in quantity, such proteins are "overexpressed" in the host cell and subsequently isolated and purified. Preinsulin for example may be produced in a recombinant prokaryotic microorganism carrying DNA encoding rat preinsulin (U.S. Pat. Nos. 4,431,740 and 4,652,525, specifically incorporated herein by reference).
Expression of multiple disulfide bond-containing eukaryotic polypeptides, and particularly mammalian proteins, in bacterial cells has frequently produced disappointing and unsatisfactory results because conditions and environment in the host cells were not conducive to correct folding. Disulfide bond formation is a process mainly restricted to proteins outside the cytoplasmic compartment such as those secreted into the lumen of the endoplasmic reticulum (ER) or the periplasm of gram negative bacteria. Correct folding may depend on the formation of cysteine-cysteine linkages and subsequent stabilization of the protein into an enzymatically active structure. However, the cytoplasm is in fact a reducing environment due to the presence of thioredoxin reductase or reduced glutathione, thus blocking oxidation so that disulfide bonds do not form. The endoplasmic reticulum (ER) apparently is more conducive to oxidation due to the presence of oxygen or oxidized glutathione.
Recent studies indicate that disulfide bond formation in vivo is a catalyzed process, whether in the ER or periplasm. In E. coli, a pathway for the formation of disulfide bonds in secreted proteins has been described, involving two proteins, DsbA and DsbB (Bardwell et al., 1993; Missiakas et al., 1993).
A role for these Dsb proteins is supported by the observation that mutants of E. coli that lack DsbA or DsbB are defective with respect to disulfide bond formation (Dailey and Berg, 1993). In the yeast Saccharomyces cerevisiae, a similar defect is found in certain mutants defective in protein disulfide isomerase (PDI) gene. Disulfide bond formation in carboxypeptidase Y in these mutants is impaired.
1.2.2 EXPRESSION OF EUKARYOTIC PROTEINS IN BACTERIAL HOSTS
It is known that disulfide bonds are critical in some proteins in order for proper folding and even in transport and secretion. Yet many proteins cannot be efficiently expressed in bacterial hosts due to failure of disulfide bond formation. Cytoplasmic expression systems in bacteria are not conducive to disulfide bond formation because of a reducing environment. The presence of proteases in the cytoplasm may cause rapid degradation of the protein, resulting in low yields.
Most exported proteins contain disulfide bonds which confer increased thermodynamic stability to the folded polypeptide chain. The sequence of events involved in cysteine oxidation and correct pairing to form native disulfide bonds is a critical step in protein folding. Due to constraints related to the reactivity or structural accessibility of cysteine thiols in proteins, disulfide bonds often form very slowly. A complex cellular machinery, whose components and mode of action are only now beginning to be understood, has evolved to catalyze these processes in vivo. In Gram-negative bacteria such as E. coli, the cytoplasm is highly reducing and therefore disulfide formation normally occurs after a polypeptide chain has been translocated across the inner membrane (Wulfing and Pluckthun, 1994; Bardwell, 1994). Genetic analysis has identified at least six genes coding for cell envelope proteins that play a role in disulfide bond formation. Four of these proteins have been characterized in some detail (Bardwell, 1994; Missiakas et al., 1995). DsbA is a 21.5 kDa enzyme having a thioredoxin-like subdomain with an extremely reactive and highly oxidizing disulfide bond but poor disulfide isomerization activity (Bardwell et al., 1991; Kamitani et al., 1992; Zapun et al., 1993; Wunderlich and Glockshuber, 1993a; 1993b; Joly and Swartz, 1994). DsbB is a cytoplasmic membrane protein which is required for the reoxidation of DsbA (Guilhot et al., 1995; Bardwell et al., 1993; Missiakas et al., 1993; Dailey and Berg, 1993). DsbC is another soluble cysteine oxidoreductase and has much higher disulfide isomerase activity than DsbA (Bardwell, 1994; Missiakas et al., 1994). Finally, the recently discovered DsbD is an inner membrane protein which has been proposed to function as a reducing source in the periplasm and to be required for maintaining proper redox conditions (Missiakas et al., 1995).
Bacterial proteins become oxidized and fold rapidly soon after export from the cytoplasm. However, the formation of native disulfide bonds in heterologous proteins with multiple cysteines is often very inefficient (Wunderlich and Glockshuber, 1993a; 1993b; De Sutter et al., 1992). Partially folded molecules are highly susceptible to degradation, thus resulting in very low yields (Wulfing and Pluckthun, 1994). The shortcomings of the disulfide bond formation machinery of E. coli with respect to eukaryotic proteins have been illuminated by analyzing the folding pathway of the Bovine PTI (BPTI) expressed in the periplasmic space (Ostermeier and Georgiou, 1994). BPTI is a 6.5-kDa protease inhibitor with three disulfide bonds. In E. coli, just as in vitro, the rate limiting step in folding is the isomerization of two disulfide intermediates. The bacterial periplasmic space is thought to be strongly oxidizing (Wunderlich and Glockshuber, 1993a; 1993b; Walker and Gilbert, 1994; Kishigami et al., 1995) and appears to lack sufficient disulfide isomerase activity required for the folding of heterologous multi-disulfide proteins. In sharp contrast to the bacterial periplasm, disulfide bond formation in eukaryotes occurs in the endoplasmic reticulum, a compartment which is maintained at relatively reducing conditions (Hwang et al., 1992). Disulfide bond formation and isomerization in the ER is catalyzed by PDI, an abundant 55-kDa enzyme which apart from its thioredoxin-like active site shares little homology with prokaryotic proteins. PDI contains two active sites that are not functionally equivalent, has been shown to both promote and inhibit protein aggregation, and can exist in different oligomerization states (Freedman et al., 1994; Lyles and Gilbert, 1994; Puig and Gilbert, 1994; Puig et al., 1994).
1.2.3 CURRENT METHODS OF PRODUCING EUKARYOTIC PROTEINS ARE INEFFICIENT
Expression enhancers for increasing yield of eukaryotic proteins expressed in E. coli cells have been reported (U.S. Pat. No. 5,336,602). The expression enhancer is simultaneously expressed with a protein of interest where the rate of expression is shown to increase by comparison with expression of the protein of interest in the absence of an enhancer. However, while yield is increased over expression when enhancer is not present, there are no indications that either correct folding is achieved or that full activity is obtained.
Eur. Pat. Appl. No. EP 510,658 describes an improvement of the yield of secreted disulfide-bonded proteins in bacterial cell by providing a simultaneous expression of a recombinant vector encoding the prokaryotic protein disulfide isomerase of E. coli and the addition of thiol reagents to the culture medium to promote correct folding of the secreted polypeptide of interest. Unfortunately, the method produced negligible secreted protein unless sufficient thiol reagent was added to the culture medium, and if too much thiol reagent was present, cells were killed and the total protein isolated declined dramatically.
tPA is one example of a pharmaceutically-important drug produced by recombinant methods. Unfortunately the current methods for producing tPA from bacterial cell culture are both costly and laborious. One such method for the production of tPA in heterologous host organisms relies on the production of inactive tPA intracellularly in inclusion bodies, and the subsequent isolation and purification of such inclusion bodies, followed by activation of the tPA once freed from the inclusion bodies. U.S. Pat. No. 5,077,392 discloses a renaturation method for refolding denatured proteins obtained after expression in inclusion bodies. tPA was isolated as a denatured reduced protein and on subsequent oxidation refolded under oxidizing conditions to obtain what was reported as up to a 26% yield of "reactivated" protein. While the method appeared to improve polypeptide yield, the process involves multiple, time-consuming steps, due to the initial recovery of the insoluble, inactive protein.
Other methods of producing tPA have employed eukaryotic cell culture methods, which are also expensive and time-consuming. Mammalian cells have been used in attempts to improve production of highly active polypeptides such as tPA. U.S. Pat. No. 4,661,453 discloses production of tPA in substantial quantities in rat prostate adenocarcinoma cells. The tPA isolated from the cell culture medium shows tPA activity, however the method is quite expensive since the mammalian cells have an origin in spontaneous adenocarcinoma cancer cells, and must be selected for the ability to produce tPA. The method has not been shown to be feasible for commercial production of proteins such as tPA on an economic scale. Even methods involving the production of tPA in recombinant chinese hamster ovary (CHO) cells, result in a cost-per-unit-dose of approximately $1200 in the current pharmaceutical market.
1.2.4 DEFICIENCIES IN THE PRIOR ART
Currently there is a lack of efficient methods of producing complex eukaryotic proteins with multiple disulfide bonds on an economic scale. Likewise, there is a need to develop methods which produce proteins that are correctly folded and active without the need for reactivation or subsequent processing once isolated from a host cell.
Therefore, what is lacking in the prior art are methods, recombinant vectors, host cells, and compositions comprising high-level expression of eukaryotic disulfide bond-containing polypeptides (such as tPA and BPTI) which are soluble, correctly-folded, active, and readily isolatable from cell extracts of prokaryotic hosts.